Epidermis/dermis equivalents and aged skin equivalents shaped therefrom

ABSTRACT

An aged skin equivalent comprising an epidermis equivalent and an aged dermis equivalent, wherein the aged dermis equivalent comprises glycated collagen. 
     An aged dermis equivalent, the epidermis equivalent obtained and methods of producing the aged skin and/or aged dermis equivalent and/or the epidermis equivalent.

This application is a divisional of application Ser. No. 09/556,124,filed on Apr. 20, 2000.

CROSS-REFERENCE TO PRIORITY APPLICATION

This application claims priority under 35 U.S.C. §119 of FR-99/04970,filed Apr. 20, 1999, hereby expressly incorporated by reference.

BACKGROUND OF THE INVENTION

1. Technical Field of the Invention

The present invention relates to novel skin equivalents, to a method forthe production thereof and to the epidermis equivalents and dermisequivalents comprising such novel skin equivalents (themselves per senovel).

2. Description of the Prior Art

For several years, the perfection of models of reconstructed skin whichpermit conducting studies required for a better understanding of therole of the skin in the mechanical domain and physiological domain hasbeen ongoing.

Thus, models which more or less approximate human skin have beendeveloped. Exemplary are the models described in EP-A-285471,EP-A-285474, EP-A-789074, EP-A-502172, EP-A-418035, WO-A-9116010,EP-A-197090, EP-A-20753, FR-A-2665175 and FR-A-2689904.

Very generally, the models of reconstructed skin described in theaforesaid publications comprise human keratinocytes associated orotherwise with other skin cells such as melanocytes and/or Langerhanscells, deposited onto a support, often a dermis equivalent, and culturedunder conditions such that they commence a program of differentiationwhich results in the formation of an epidermis equivalent.

The dermis equivalents described to date are either artificial membranessuch as, for example, Millipore filters, collagen-based subcutaneoussubstitutes, plastic or any other support which is compatible with cellviability, or supports which are more developed such as to approximatenatural dermis, for example previously de-epidermilized dermis orCollagen/fibroblast mixed lattices.

In the collagen/fibroblast mixed lattices the association of nativecollagen and isolated human fibroblasts ultimately provides a dermisequivalent which mimics a dermis which has not been subjected to theactions of the weather.

The protocols employed for the preparation of said lattices use collagenoriginating in general from young tissues, this collagen having,however, been subjected to the important post-translationalmodifications which intervene in the complex but nevertheless normalprocesses of its biosynthesis, but not having undergone all of themodifications which can intervene, in particular, during aging.

It is known to this art, in particular, to this art, that during agingand during the progress of certain diseases such as diabetes,non-enzymatic processes operate which involve an ose (glucose or ribose)which reacts according to the Maillard reaction with an amino group (forexample a lysine residue) of the collagen to form a Schiff's base. Thisbase, after undergoing an Amadori molecular rearrangement, may provide,by a succession of reactions, intramolecular bridging such as, forexample, of pentosidine type. This phenomenon, termed glycation ofcollagen, increases uniformly with age, leading to a uniform increase inthe glycation-product content of the skin. These glycation productsinclude, for example, pyrraline, carboxymethyl-lysine, pentosidine,crosslines, N^(e)-(2-carboxyethyl)lysine (CEL), glyoxal-lysine dimer(GOLD), methylglyoxal-lysine dimer (MOLD), 3DG-ARG imidazolone,versperlysines A, B, C, threosidine or, alternatively, advancedglycosylation end products or AGEs. This phenomenon is amplified incertain disease states, such as, for example, diabetes.

Without wishing to be bound to any particular theory as regards aging ofthe skin, it should be noted that other characteristics which might alsobe a consequence of these glycation phenomena, such as a decrease inheat denaturation, an increase in resistance to enzymatic digestion andan increase in intermolecular bridging, have been demonstrated duringaging of the skin (Tanaka S. et al., 1988, J. Mol. Biol., 203, 495-505;Takahashi M. et al., 1995, Analytical Biochemistry, 232, 158-162). Inaddition, modifications due to the glycation of certain constituents ofthe basal membrane such as collagen IV, laminine and fibronectin havebeen demonstrated (Tarsio J F. et al., 1985, Diabetes, 34, 477-484;Tarsio J F. et al., 1988, Diabetes, 37, 532-539; Sternberg M. et al.,1995, C. R. Soc. Biol., 189, 967-985).

Thus, it is understood that during aging of the skin the physicochemicalproperties of collagen are modified and such collagen becomes moredifficult to dissolve and more difficult to degrade.

Thus, one of the components of aged skin indeed appears to be glycatedcollagen.

It is also very well known that the skin constitutes a close associationbetween at least two components thereof, i.e., the epidermis and thedermis. The interactions between the dermis and the eipdermis are suchthat it is reasonable to consider that a modification of one may haveconsequences on the other. It may be suspected that the aging of thedermis, in particular with its glycation phenomena, can only haveconsequences for the epidermis to which it is associated. Thus, duringskin aging, the glycation of the collagen must promote modifications ofthe epidermis which must participate in the aging of the epidermis.

In this respect, it has now been demonstrated that a constitutiveprotein of normal epidermis, i.e., the β1 type integrin, anextracellular matrix receptor (see Ruoslahti E., 1991, Cell Bioloay ofExtracellular Matrix, Plenum press New York, 343363), shows adistribution of its expression in aged epidermis which is very differentfrom that in young epidermis. Specifically, while in a young epidermis,i.e., for the purposes of this invention an epidermis from a youngsubject, this protein is expressed in the very deep layers of theepidermis, i.e., up to a maximum of the second suprabasal layer, thesituation is completely different in an aged epidermis, i.e., for thepurposes of this invention in an epidermis from an elderly subject,where this protein is expressed in most layers of the epidermis,directly through to the last suprabasal layers under the cornifiedlayer.

To date, no model of reconstructed skin in vitro is capable, eitherbecause of the protocols of preparation technique, or the simple factthat once reconstituted it does not undergo modification, of producing askin equivalent at least one of the constituents of which provides oneof the components of skin aging. Thus, no model of reconstructed skin invitro presents the properties of an aged skin or permits the study ofthe processes resulting therein, or the study of the compounds and/orcompositions which would at least make it possible to slow down orretard this/these process(es). The only known evaluations of thesephenomena entail in vivo studies, either in animals or humans. Mostparticularly, for ethical reasons, the advantage of having such a modelis conspicuously apparent.

Studies on glycation are known in the prior art. For example, a methodis described for obtaining a connective tissue equivalent in the form ofa lattice of glycated collagen and fibroblasts (see in this respect Freyet al. (1992, C. R. Soc. Biol., 187, 223-231). However, not only haveFrey et al. never investigated or even suggested the possibility ofpreparing a skin equivalent from their lattice, they have never comparedthis lattice to any dermis equivalent. In addition, but whilerecognizing the validity of the conjunctive tissue model of Frey et al.,it will be appreciated that the protocol employed cannot provide theobjectives established hereby, i.e., to reproduce in vitro a skin, andconsequently an epidermis and a dermis, which has all or part of theproperties of an aged skin.

By incubating the collagen and sugar for 9 hours at a temperature of 4°C., Frey et al. initiate the collagen glycation reaction, this reactionthen continuing in the lattice in which the collagen is in this mannerpreglycated. If it is desired to attain a sufficient level of glycation,such as to mimic an aged skin, it is then necessary to permit theglycation reaction to continue in the lattice formed in this fashion,i.e., in the process of contraction, for a further time which issufficient to attain the desired level. However, those skilled in thisart are cognizant that to establish an epidermis equivalent containingat least keratinocytes on a dermis equivalent of collagen/fibroblastlattice type, the seeding of the keratinocytes must be conducted onto alattice which cannot have exceeded a determined stage of contraction. Itwill thus be seen, therefore, that it is not possible to obtain by theFrey protocol a dermis equivalent which mimics an aged or very ageddermis, i.e., highly or very highly glycated.

SUMMARY OF THE INVENTION

Accordingly, a major object of the present invention is the provision ofa skin equivalent which comprises an epidermis equivalent produced on anaged dermis equivalent, particularly on a lattice comprising at leastglycated collagen and fibroblasts.

A significant advantage of this invention is providing a model ofreconstructed skin in which at least one of the components thereof wouldhave one of the aspects of an aged skin.

DETAILED DESCRIPTION OF BEST MODE AND SPECIFIC/PREFERRED EMBODIMENTS OFTHE INVENTION

More particularly according to the present invention, the assigneehereof has long been interested in the production of in vitro models ofreconstructed skin, and a novel model of reconstructed skin has now beendeveloped comprising an epidermis equivalent and a dermis equivalent,said dermis equivalent comprising an aged dermis equivalent,particularly a lattice which comprises at least glycated collagen andfibroblasts.

Thus, the present invention features a novel aged dermis equivalent,comprising at least glycated collagen and fibroblasts.

Different techniques for tracking the formation of the glycationproducts are described in the prior art. In this respect, methods whichare representative are described by Cefalu W. T. et al. (Journal ofGerontology; Biological Sciences, 1995, vol. 50 A, No. 6, 13337-13341),by Sell D. R. (Diabetes/Metabolism, 1991, vol. 7, No. 4, 239-251), byMiyata T. et al. (Journal of the American Society of Nephrology, 1996,vol. 7, No. 8, 1198-1206), or, alternatively, by Furth A. J. (AnalyticalBiochemistry, 1991, 192, 109-111).

Thus, it is possible to measure the level of glycating compound bound tothe collagen and/or the level of glycating compound remaining after thereaction. As described above, the glycation of collagen leads to theformation of glycation products such as, for example, pyrraline,carboxymethyl-lysine, pentosidine, crosslines,N^(e)-(2-carboxyethyl)lysine (CEL), glyoxal-lysine dimer (GOLD),methylglyoxal-lysine dimer (MOLD), 3DG-ARG imidazolone, versperlysinesA, B and C, threosidine or alternatively advanced glycosylation endproducts or AGEs.

Certain of these glycation products have the particular feature ofemitting a measurable fluorescence after excitation. For example,pentosidine, when excited at a wavelength (λex) of 328 nm, emits afluorescence at a wavelength (λem) of 378 nm. Similarly, AGEs, whenexcited at a wavelength (λex) of 370 nm, emit a fluorescence at awavelength (λem) of 440 nm. The ratio of the fluorescence which isemitted by a given glycation product in the dermis equivalent comprisingat least glycated collagen and fibroblasts to the fluorescence which isemitted by the same glycation product in a control dermis equivalentcomprising at least non-glycated collagen and fibroblasts, measuredunder the same experimental conditions, makes it possible tocharacterize the level of glycation of the aged dermis equivalent.

Particularly, according to the invention, the fluorescence of thepentosidine and/or the AGEs is measured both in the aged dermisequivalent comprising. glycated collagen and in a control comprisingnon-glycated collagen.

Thus, according to the invention, the aged dermis equivalent has a levelof glycation ranging from 2 to 30 and particularly from 8 to 18.

Consistent herewith, the collagen may be any type of collagen of anyorigin. In this respect, reference is made to the different types ofcollagen noted in the reviews of Van der Rest and Garonne, 1990,Biochem, vol. 72, 473-484 or, alternatively, 1991, Faseb Journal, vol.5, 2814-2823. Thus, according to the invention, the collagen ispreferably selected from among the fibrillary collagens of type I, IIIor V.

Preferably, collagen of animal origin is used, particularly collagen ofbovine origin.

The preferred collagen according to the invention is type I collagen.Very preferably, type I bovine collagen is employed.

It will of course be appreciated that a mixture of collagen of differenttypes in any proportion and/or of different origins may be used.

Also according to the invention, the fibroblasts may emanate from anyorigin, but are preferably fibroblasts of human origin. They may beprepared according to any known technique of the prior art, for exampleby mechanical and/or enzymatic dissociation of the macromolecules of theextracellular matrix of the dermis or by growth of fibroblasts fromexplants.

Too, the dermis equivalent of the invention comprises at least glycatedcollagen and fibroblasts, but may also contain any other component thatmight be advantageous to introduce therein such as, for example,endothelial cells, macrophages or alternatively nerve cells.

The present invention also features an epidermis equivalent comprisingat least keratinocytes, characterized in that it can be formed byseeding at least keratinocytes onto a dermis equivalent comprising atleast glycated collagen and fibroblasts.

As above indicated, depending on age, certain markers of the eipdermiscould undergo modifications either in their quantity or in thelocalization of their expression. Particularly, it has been shown thatthe expression of β1 integrin in the epidermis is modified in itslocalization with the age of the epidermis. Specifically, while in ayoung epidermis this protein is expressed strictly in a maximum of thefirst two suprabasal layers, with age this expression, while conservingits localization in the first two suprabasal layers, appears in layerswhich are more and more superficial to the point of appearing in all ofthe suprabasal layers including the final layers adjoining the stratumcorneum.

Thus, in one embodiment of the invention the epidermis equivalent ischaracterized in that it has modified expression of β1 integrin,particularly expression of β1 integrin in the cells of at least thefirst three suprabasal layers.

Very preferably, according to the invention, the epidermis equivalentcomprising at least keratinocytes is characterized in that it hasmodified expression of β1 integrin, particularly expression of β1integrin in the cells of at least the first three suprabasal layers, andin that it is obtained by seeding at least keratinocytes onto a dermisequivalent comprising at least glycated collagen and fibroblasts.

Of course, any method which makes it possible to demonstrate theexpression of β1 integrin can be used to characterize the epidermis ofthe invention. Labelling with the aid of anti-β1 integrin antibodies isexemplary.

The keratinocytes used according to the invention may emanate from anyorigin, but are preferably keratinocytes of human origin. They may beprepared according to any known method of the prior art. Culturing fromdissociated epidermis originating from a normal skin sample, or theculturing of keratinocytes derived from the sheath of the hairfollicles, are representative examples.

Preferably, keratinocytes from normal human skin are used.

Also, preferably, the keratinocytes are prepared from dissociated humanepidermis originating from a normal human skin sample according to thetechnique described in Régnier et al., Frontier of Matrix Bioloay, Vol.9, 4-35 (Karger, Basle 1981).

The epidermis equivalent of the invention comprises at leastkeratinocytes, but it may comprise any other cell type which might beincorporated therein such as, for example, Langerhans cells and/orprecursors of Langerhans cells and/or melanocytes.

Of course, the skin equivalent which has the best similarity with normalskin is the skin equivalent which contains the three essential celltypes present in normal skin.

Thus, advantageously the model of reconstructed skin according to theinvention comprises, in addition, melanocytes and/or Langerhans cellsand/or precursors of Langerhans cells.

The melanocytes according to the invention may be isolated from anyorgan which contains them such as, for example, normal skin or hairfollicles.

Preferably, melanocytes isolated from normal skin are used.

Any known method of preparation of melanocytes of the prior art may beused according to the invention. The method described in Olsson et al.,Acta Derm. Venereol., 1994, 74, 226-268, is one such example.

The Langerhans cells and/or the precursors of Langerhans cells which canbe employed according to the invention may be as described in EuropeanPatent Application published under the number EP-A-789074, assigned tothe assignee hereof.

This invention also features an aged skin equivalent, which comprises atleast one epidermis equivalent and one aged dermis equivalent.

In another embodiment of the invention, the aged skin equivalentcomprises an aged dermis equivalent as described above.

In another embodiment of the invention, the aged skin equivalentcomprises an epidermis equivalent as described above.

An aged skin equivalent which is very preferable according to theinvention comprises an epidermis equivalent comprising at leastkeratinocytes which have modified expression of β1 integrin,particularly expression of β1 integrin in the cells of at least thefirst three suprabasal layers and is characterized in that it can beobtained by seeding at least keratinocytes onto an aged dermisequivalent comprising at least glycated collagen and fibroblasts, saidaged dermis equivalent having a level of glycation ranging from 2 to 30,and particularly from 8 to 18.

The present invention also features a method for preparing an aged skinequivalent comprising an epidermis equivalent and a dermis equivalentwhich itself comprises a lattice comprising at least glycated collagenand fibroblasts, wherein a first step a lattice comprising at leastglycated collagen and fibroblast is prepared and in a second step anepidermis equivalent comprising at least keratinocytes is reconstitutedon the lattice obtained in the first step.

The first step may be carried out by any known technique of the priorart, provided that the collagen may be glycated either beforehand,during or after the formation of the lattice. Preferably, according tothe invention, a lattice is prepared in which either collagen which isglycated prior to the formation of the lattice is used or a glycationagent is added to the mixture of collagen and fibroblast which is used,in order to effect the glycation either during the preparation of thelattice or after formation of the lattice.

Preferably, the lattice is prepared using previously glycated collagen.

Also preferably, the lattice is prepared according to the methoddescribed by Asselineau et al., 1987, (Models in Dermato, vol. III, Ed.Lowe & Maibach, 1-7) using preglycated collagen.

Any known method of glycation may be used to produce glycated collagen.For example, the methods described by Tanaka et al. (J. Mol. Biol.,1988, 203, 495-505), Tarsio J F. et al. (1985, Diabetes, 34, 477-484),Tarsio J F. et al. (1988, Diabetes, 37, 532-539) or, alternatively, byFrey J. et al. (1992, C. R. Soc. Biol., 187, 223-231) are exemplarythereof.

Preferably, the glycation may be carried out by contacting a solution ofat least one collagen with a solution of at least one glycating agent insuch manner as to induce the glycation reaction of the collagen in vitroin the absence of cells.

As indicated above, the collagen may be any type of collagen, of anyorigin, alone or in admixture.

Preferably, the collagen is of animal origin, particularly collagen ofbovine origin.

The preferred collagen according to the invention is type I collagen.Very preferably, type I bovine collagen is employed.

The collagen solution is advantageously at a concentration of from 2mg/ml to 6 mg/ml and preferably from 3 mg/ml to 5 mg/ml.

The glycating agent may be any agent which enables the glycation, i.e.,which is capable of reacting according to the Maillard reaction with anamino group of the collagen to form a Schiff's base. In this respect,exemplary intermediates of the Maillard reaction include for example,glucosone, 3-deoxyglucosone, glyoxal, methylglyoxal or, alternatively,sugars.

Any type of sugar can be used according to the invention, whether it isin monomeric or polymeric form. According to the invention, a monomericsugar is preferred.

By “sugar” are intended compounds which possess several alcohol groupsand at least one aldehyde group. Oses are particularly representativethereof.

Among the sugars which are suitable according to the invention, ribose,fructose or glucose, inter alia, are exemplary. Preferably, ribose orglucose is used.

The sugar may be in any one of the dextrorotatory or laevorotatoryconformations. Preferably, a sugar in dextrorotatory conformation isused.

Particularly, according to the invention D-fructose, D-ribose orD-glucose is used. Preferably D-ribose or D-glucose is used.

The glycating agent may be used either alone or in admixture.

The amount of glycating agent which can be used according to theinvention should be sufficient to permit initiating the non-enzymaticreactions which lead to the formation of Schiff's base. It should beunderstood that varying this amount makes it possible to obtain a finalproduct, the glycated collagen, whose level of glycation varies fromrelatively unglycated to very highly glycated. Thus, the amount ofglycating agent advantageously ranges from 0.5% to 20%, preferably from1% to 10% by weight of the total weight of the collagen solution.

The glycation reaction is carried out at a temperature which is close toroom temperature. Thus, the reaction is carried out at a temperatureranging from 15° C. to 30° C., preferably from 20° C. to 25° C.

The duration of the glycation reaction depends on the desired level ofglycation. It will be appreciated that the longer the time the higherthe level of glycation. Thus, the duration of the glycation reactionadvantageously ranges from 15 days to 2 months, preferably from 25 daysto 35 days.

When it is determined to carry out the glycation prior to thepreparation of the lattice, it is possible to subject the glycatedcollagen to all the subsequent steps necessary for producing the purestpossible product. Thus, it is possible to attempt to eliminate any traceof glycating agent which would not have reacted during the reaction. Forthis any known technique may be employed. For example, the preglycatedcollagen solution is subjected to a series of dialyses against waterand/or acetic acid.

In another embodiment of the invention, the “preglycated” collagensolution obtained may be mixed with native collagen before use for thepreparation of the dermis equivalent. In this instance, the ratio ofglycated collagen to non-glycated collagen may range from 25 to 75 andpreferably from 45 to 55. Variation of this ratio makes it possible tomodulate the level of glycation of the lattice. Preferably, according tothe invention a mixture of glycated collagen and non-glycated collagenis used, and even more preferably a mixture of glycated collagen andnon-glycated collagen in a 50/50 ratio.

The second step of the method of the invention may be carried out by anyknown technique of the prior art.

In this respect, techniques which are representative are described inEP-A-285471, EP-A-285474, EP-A-789074, EP-A-502172, EP-A-418035,WO-A-9116010, EP-A-197090, EP-A-20753, FR-A-2665175 and FR-A-2689904,or, alternatively, that described by Asselineau et al., 1985, (Exp. Cel.Res., 536-539) and 1987, (Models in Dermato., vol. III, Ed. Lowe &Maibach, 1-7).

Preferably, the method described by Asselineau and colleague isemployed.

The keratinocytes according to the invention may emanate from anyorigin, but are preferably keratinocytes of human origin. They may beprepared according to any known procedure of the prior art. Culture fromdissociated epidermis originating from a normal skin sample, or theculture of keratinocytes derived from the sheath of hair follicles, arerepresentative.

Preferably, the keratinocytes are prepared from dissociated humanepidermis originating from a normal human skin sample according to themethod described in Regnier et al., Frontier of Matrix Biology, Vol. 9,4-35 (Karger, Basle 1981).

Advantageously, after seeding the keratinocytes onto the support, theculture may be maintained submerged in a nutritive medium which may be,for example, the medium described by Rheinwald and Green, 1975, (Cell,6, (3), 317-330), this medium allowing the proliferation of thekeratinocytes (referred to hereinafter as medium 3F).

After an incubation time of 3 to 15 days, preferably of 7 to 9 days, theskin equivalent is maintained at the air/liquid interface by, forexample, depositing it onto a metal grid. The liquid then preferablyconsists of the same nutritive medium as the previous one.

The incubation then continues until production of a skin equivalenthaving the properties of a skin, i.e., the support on which is anepidermis equivalent having the four conventional types of cell layer,i.e., the basal, suprabasal, granular and cornified layers.

Thus, the incubation continues for a duration advantageously rangingfrom 5 days to 30 days, preferably from 7 days to 10 days.

The model of reconstructed skin produced in this manner comprises twoentities: the support and the epidermis equivalent, that it is possibleto physically separate from each other.

The epidermis equivalent may thus be used separately from the support.

As above indicated, to date no in vitro model of reconstructed skin hadthe properties of an aged skin or permitted the study of the processesresulting therein or the study of the compounds and/or compositionswhich would at least make it possible to modify the process thereof. Theskin equivalent obtained according to the invention resolves theseproblems, since it has at least one of the properties of aged skin,i.e., a glycated collagen. This important property which the model andmethod thereof make it possible to vary in any proportion, permits thestudy of the glycation phenomenon in itself and modulators (inhibitorsor activators) of this phenomenon, the study of phenomena linked to agedskin such as, for example, wrinkles, the study of modulators (isolatedcompounds and/or compositions) of the appearance of the wrinkles(particularly the inhibitors), the study of photoaging and the effect onthe skin of ultraviolet rays, as well as modulators of these effects(protective compounds and/or compositions, filters, etc.), the study ofthe influence of glycation on the components of the skin (bristlesand/or hair, blood vessels, nerve fibers, etc.), and in the therapeuticdomain the study of the complications caused by diabetes via glycation.

Thus, this invention also features employing an aged skin and/or ageddermis equivalent and/or an aged epidermis equivalent as described abovefor the study of the glycation phenomenon itself and modulators(inhibitors or activators) of this phenomenon, the study of phenomenalinked to aged skin such as, for example, wrinkles, the study ofmodulators (isolated compounds and/or compositions) of the appearance ofthe wrinkles (particularly the inhibitors), the study of photoaging andthe effect on the skin of ultraviolet rays as well as modulators ofthese effects (protective compounds and/or compositions, screeningagents, etc.), the study of the influence of glycation on the componentsof the skin (bristles and/or hair, blood vessels, nerve fibers, etc.),and in the therapeutic domain the study of the complications caused bydiabetes via glycation.

It will also been seen that, according to the invention, the epidermisequivalent which is reconstructed on the dermis equivalent comprising atleast glycated collagen and fibroblasts may have a modified distributionof the expression of β1 integrin. In this instance, if the variation inthe distribution of this marker is linked to the age of the epidermis,it is conceivable to use the aged skin equivalent of the invention toevaluate any product able to treat aged skin by measuring its effect bythe effect that it produces on the modification of the distribution ofβ1 integrin expression in the epidermis equivalent.

FIG. 1 better illustrates the invention, without however limiting itsscope. In this figure, the photographs show sections of skin equivalentsafter immunolabelling with the aid of an anti-β1 integrin antibody.Photo 1 represents the immunolabelling of a skin equivalent comprisingan epidermis equivalent reconstructed on a dermis equivalent which isprepared with non-glycated collagen. Photo 2 represents theimmunolabelling of a skin equivalent comprising an epidermis equivalentreconstructed on a dermis equivalent which is prepared with glycatedcollagen according to the invention.

In order to further illustrate the present invention and the advantagesthereof, the following specific examples are given, it being understoodthat same are intended only as illustrative and in nowise limitative.

EXAMPLES

Media and buffers unless otherwise indicated; all media and buffers usedin the following examples are described in Bell et al., 1979, (P.N.A.S.USA, 76, 1274-1278), Asselineau and Prunieras, 1984, (British J. ofDerm., 111, 219-222) or Asselineau et al., 1987, (Models in Dermato.,vol. III, Ed. Lowe & Maibach, 1-7).

Example 1 Preparation of Glycated Bovine Collagen I

10 ml of bovine collagen I solution at a concentration of 3 mg/ml, 0.8ml of 0.5N sodium hydroxide to neutralize the acidic collagen solutionand 100 μl of a 1M solution of D-ribose in water were placed into a 50ml Falcon tube.

The tube, rendered opaque to the light, was placed horizontally andgently shaken at room temperature (25° C.) for 1 month.

At the end of the “preglycation”, the solution was placed in dialysistubing (Spectra/Poly labo 32 mm No. 132655/85716) and was subjected to aseries of successive dialyses:

24 hours against demineralized water at 4° C. to eliminate the unboundsugar or the collagen degradation products;

7 days against 0.5N acetic acid to redissolve the collagen, in 2 bathsof three and a half days;

3×24 h against 0.017N acetic acid with bath changing every day.

After the last dialysis, the contents of the dialysis tubing wererecovered in a sterile beaker and the solution was transferred into asterile 50 ml Falcon tube, rendered opaque to the light.

The “preglycated” collagen solution was then ready to use. It may bestored at 4° C.

Example 2 Preparation of an Aged Dermis Equivalent

3.22 ml of 1.76×MEM medium, 0.63 ml of foetal calf serum, 0.35 ml of0.1N sodium hydroxide and 0.20 ml of an MEM medium/Hepes mixturecontaining 10% foetal calf serum (MEM/Hepes/FCS10) were placed into asterile Falcon tube.

0.50 ml of MEM medium/Hepes/FCS10 were then added containing fibroblastsderived from human mammary plastic surgery which were preparedbeforehand according to the technique described by Bell et al., 1979,(P.N.A.S. USA, 76, 1274-1278), Asselineau and Prunieras, 1984, (BritishJ. of Derm., 111, 219-222) or Asselineau et al., 1987, (Models inDermato., vol. III, Ed. Lowe & Maibach, 1-7), at a concentration of1×10⁶ cells for 0.5 ml of culture medium.

2 ml of a volume/volume mixture of preglycated collagen from Example 1and non-glycated collagen having served in the preparation of thepreglycated collagen from Example 1, at a concentration of 3 mg/ml inacetic acid at 1/1000, was then slowly added, against the wall of thetube, in such manner as to observe the appearance of a whiteish cloud.The entire medium was then mixed cautiously and plated in a 60 mmdiameter Petri dish (type Falcon 60 mm, ref. 1016). The Petri dish wasthen placed in an incubator at 37° C. and left for about 2 hours, 30minutes. When the appearance of 2 phases (gel+medium) was observed, thelattice was cautiously dissociated from its support and the latticedissociated from its support in this manner was left in the incubatorfor 4 days.

Example 3 Measurement of the Level of Glycation of the Aged DermisEquivalent From Example 2

In parallel to the production of the aged dermis equivalent of Example2, a dermis equivalent without glycated collagen (but with non-glycatedcollagen) was produced. This equivalent was used as a control in thedetermination of the level of glycation of the glycated dermisequivalent from Example 2.

Two lattices (one aged, one control) prepared according to Example 2were rinsed three times in phosphate buffered saline (PBS), then dried.The lattices were then placed in an Eppendorf tube and subjected todigestion with pepsin (Sigma P-6887) at 37° C. in a water bath overnight(12 hours) in a proportion of 500 μg of pepsin per lattice in 0.5 ml of0.5N acetic acid.

515 μl of 0.5N sodium hydroxide were then added to each tube and thecontents of each tube were filtered through a 0.22 μm spin filter(Sigma).

The fluorescence was then measured with the aid of a Hitachispectrofluorimeter, model F2000. The fluorescence emitted by pentosidineat λem=378 nm after excitation at λex=328 nm and the fluorescenceemitted by AGEs at λem=440 nm after excitation at λex=370 nm were thusmeasured.

The results obtained are reported in the Table below:

TABLE Pentosidine AGEs Control 650 430 Glycated lattice 6100 1820

In this example the level of glycation of the aged dermis equivalent wasthus established at 9.4 for the pentosidine and at 4 for the AGEs.

Example 4 Preparation of an Aged Skin Equivalent

An aged dermis equivalent as prepared in Example 2 was set up and wellspread out in a Corning θ 60 mm culture dish on a droplet of collagen“glue” (0.6 ml), then maintained at 37° C. in an incubator for 20-30minutes.

A sterile steel ring was placed on the lattice and 0.5 ml of a cellularsuspension of human keratinocytes originating from mammary plasticsurgery which were prepared according to Régnier et al., (Frontier ofMatrix Biology, Vol. 9, 4-35, Karger, Basle 1981), in a proportion of100,000 cells/ml in MEM medium 10% FCS+3F, were placed inside the ring.About 6 ml of medium (MEM 10% FCS+3F) were placed around the ring andthe dish was placed in an incubator at 37° C. for 2 hours. The ring wasthen removed and the dish again placed in the incubator.

After 8 days, the culture was then placed at the air/liquid interface,said liquid consisting of the same medium as above.

The culturing was then continued for 1 week until production of anepidermis equivalent which was histologically satisfactory, i.e., anepidermis equivalent which had the four conventional cell layers, i.e.,the basal, suprabasal, granular and cornified layers.

Example 5 Characterization of the Expression of β1 Integrin in theEpidermis Equivalent Obtained in Example 4

The expression of β1 integrin in the epidermis equivalent obtained inExample 4 was observed after immunolabelling with the aid of a mousemonoclonal antibody directed against β1 integrin (Immunotech, Marseille,France, Cat. 1151): After freezing, the skin equivalents obtained inExample 4 were sectioned into 5 μm thick slices with the aid of acryostat (make/model). The sections were then rinsed twice with PBS and25 μl of anti-β1 integrin antibody diluted at 1/50 (Immunotech,Marseille, France, Cat5.1151) were deposited onto each section and leftfor 30 minutes at room temperature (25° C.). The sections were thenrinsed twice with PBS and 25 μl of FITC conjugated antibody (rabbit antimouse FITC, Dako F232) were deposited onto each section and left for 30minutes at room temperature (25° C.). The sections were rinsed twicewith PBS and observed, after mounting, under a LEICA fluorescencemicroscope, model LEITZ DMRB.

Observation indicated that in the control the β1 integrin was expressedin the basal layer of the epidermis reconstructed on the non-glycateddermis equivalent and in the first suprabasal layer (FIG. 1, photo 1),whereas it was expressed in all of the suprabasal layers, up to justunder the stratum corneum, in the epidermis reconstructed on the ageddermis equivalent (FIG. 1, photo 2).

These results correlate with the observation of a distribution in vivoof the expression of the β1 integrin in the basal layer and firstsuprabasal layer in a young subject's skin, and in the basal layer andat least the first 3 suprabasal layers in the skin of an elderlyindividual.

While the invention has been described in terms of various preferredembodiments, the skilled artisan will appreciate that variousmodifications, substitutions, omissions, and changes may be made withoutdeparting from the spirit thereof. Accordingly, it is intended that thescope of the present invention be limited solely by the scope of thefollowing claims, including equivalents thereof.

What is claimed is:
 1. Aged skin equivalent, comprising at least oneepidermis equivalent and one aged dermis equivalent wherein the ageddermis equivalent comprises at least glycated collagen and fibroblastsand wherein the epidermis equivalent comprises at least keratinocytes,said epidermis equivalent having a modified distribution of expressionof β1 integrin.
 2. Aged skin equivalent according to claim 1, whereinthe epidermis equivalent is obtained by seeding of at leastkeratinocytes onto a dermis equivalent comprising at least glycatedcollagen and fibroblasts.
 3. Aged skin equivalent according to claim 1,wherein the modified distribution of β1 integrin is expression of β1integrin in the cells of at least the first three suprabasal layers. 4.Aged skin equivalent according to claim 1, wherein the epidermisequivalent has a modified distribution of expression of β1 integrin andis obtained by seeding of at least keratinocytes onto a dermisequivalent comprising at least glycated collagen and fibroblasts. 5.Aged skin equivalent according to claim 1, wherein the epidermisequivalent has β1 integrin expression in the cells of at least the firstthree suprabasal layers, said epidermis equivalent being obtained byseeding of at least keratinocytes onto a dermis equivalent comprising atleast glycated collagen and fibroblasts.
 6. A method for preparing anaged skin equivalent comprising an epidermis equivalent and an ageddermis equivalent, the method comprising (1) preparing an aged dermisequivalent comprising a lattice of at least glycated collagenfibroblasts; and (2) reconstructing an epidermis equivalent comprisingat least keratinocytes onto the lattice of step 1; wherein the epidermisequivalent has a modified distribution of β1 integrin expression.
 7. Amethod according to claim 6, further comprising glycating the collagenused in the first step prior to preparing the lattice.
 8. A methodaccording to claim 7, comprising glycating the collagen by contacting asolution of at least one collagen and a solution of at least oneglycating agent in such manner as to induce the glycation reaction invitro in the absence of cells.
 9. A method according to claim 6, whereinthe collagen comprises collagen of human or animal origin.
 10. A methodaccording to claim 7, wherein the collagen comprises collagen of humanor animal origin.
 11. A method according to claim 8, wherein thecollagen comprises collagen of human or animal origin.
 12. A methodaccording to claim 9, wherein the collagen comprises collagen of animalorigin.
 13. A method according to claim 10, wherein the collagencomprises collagen of animal origin.
 14. A method according to claim 11,wherein the collagen comprises collagen of animal origin.
 15. A methodaccording to claim 12, wherein the collagen comprises collagen of bovineorigin.
 16. A method according to claim 13, wherein the collagencomprises collagen of bovine origin.
 17. A method according to claim 14,wherein the collagen comprises collagen of bovine origin.
 18. A methodaccording to claim 6, wherein the collagen comprises type I, III or Vcollagen.
 19. A method according to claim 9, wherein the collagencomprises type I, III or V collagen.
 20. A method according to claim 15,wherein the collagen comprises type I, III or V collagen.
 21. A methodaccording to claim 18, wherein the collagen comprises type I collagen.22. A method according to claim 19, wherein the collagen comprises typeI collagen.
 23. A method according to claim 20, wherein the collagencomprises type I collagen.
 24. A method according to claim 21, whereinthe collagen comprises bovine type I collagen.
 25. A method according toclaim 22, wherein the collagen comprises bovine type I collagen.
 26. Amethod according to claim 23, wherein the collagen comprises bovine typeI collagen.
 27. A method according to claim 6, wherein the collagen isat a concentration of from 2 mg/ml to 6 mg/ml.
 28. A method according toclaim 27, wherein the collagen is at a concentration of from 3 mg/ml to5 mg/ml.
 29. A method according to claim 27, wherein the collagen isbovine type I collagen.
 30. A method according to claim 28, wherein thecollagen is bovine type I collagen.
 31. A method according to claim 8,wherein the glycating agent is an agent which with an amino group of thecollagen to form a Schiff's base according to the Maillard reaction. 32.A method according to claim 31, wherein the glycating agent comprisesglucosone, 3-deoxyglucosone, glyoxal, methylglyoxal or a sugar.
 33. Amethod according to claim 32, wherein the glycating agent is a sugar.34. A method according to claim 33, wherein the sugar comprises an ose.35. A method according to claim 34, wherein the ose comprises ribose,fructose or glucose.
 36. A method according to claim 8, wherein theglycating agent comprises ribose or glucose.
 37. A method according toclaim 8, wherein the amount of glycating agent is from 0.5% to 20% byweight of the total weight of the collagen solution.
 38. A methodaccording to claim 37, wherein the amount of glycating agent is from 1%to 10% by weight of the total weight of the collagen solution.
 39. Amethod according to claim 7, wherein the glycation reaction is carriedout at a temperature of from 15° C. to 30° C.
 40. A method according toclaim 8, wherein the glycation reaction is carried out at a temperatureof from 15° C. to 30° C.
 41. A method according to claim 39, wherein theglycation reaction is carried out at a temperature of from 20° C. to 25°C.
 42. A method according to claim 7, wherein the duration of theglycation reaction is from 15 days to 2 months.
 43. A method accordingto claim 8, wherein the duration of the glycation reaction is from 15days to 2 months.
 44. A method according to claim 42, wherein theduration of the glycation reaction is from 25 days to 35 days.
 45. Amethod according to claim 43, wherein the duration of the glycationreaction is from 25 days to 35 days.
 46. A method according to claim 6,wherein the lattice further comprises non-glycated collagen.
 47. Amethod according to claim 6, wherein the keratinocytes comprisekeratinocytes of human origin.
 48. A method according to claim 7,wherein the keratinocytes comprise keratinocytes of human origin.
 49. Amethod according to claim 8, wherein the keratinocytes comprisekeratinocytes of human origin.